Solid-state NMR structural measurements on the membrane-associated influenza fusion protein ectodomain

The 185 residue "FHA2" domain of the influenza virus hemagglutinin protein lies outside the virus and plays a significant role in catalyzing fusion between viral and host cell membranes. FHA2 was expressed in Escherichia coli with a yield of 3 mg/L of fermentation and after purification was shown to be folded in detergent and to rapidly catalyze vesicle fusion. More than half of the FHA2 residues are the first residue in a unique sequential pair, and for a particular pair, (CO)-C-13 and N-15 amino acid type labeling was, respectively, done for the first and second residues in the pair. For membrane-associated FHA2, NMR filtering allowed selective observation of the 13CO signal of the first residue, and the 13CO chemical shift was correlated with local secondary structure. Such shift/conformational measurements were made at critical glycine residues in the functionally important "fusion peptide" region of FHA2 and at a more C-terminal leucine. This should be a general approach to mapping the conformation of membrane-associated FHA2 and for testing FHA2 structure-function models. The approach will also be applicable to other viral fusion proteins and other large membrane proteins.